Slow inactivation does not affect movement of the fast inactivation gate in voltage-gated Na+ channels.

Vedantham V, Cannon SC - J. Gen. Physiol. (1998)

Bottom Line:
In this study, we probed this relationship by examining the effects of slow inactivation on a conformational marker for fast inactivation, the accessibility of a site on the Na+ channel III-IV linker that is believed to form a part of the fast inactivation particle.We found that burial of cys1304 is not altered by the onset of slow inactivation, and that recovery of accessibility of cys1304 is not slowed after long (2-10 s) depolarizations.These results suggest that (a) fast and slow inactivation are structurally distinct processes that are not tightly coupled, (b) fast and slow inactivation are not mutually exclusive processes (i.e., sodium channels may be fast- and slow-inactivated simultaneously), and (c) after long depolarizations, recovery from fast inactivation precedes recovery from slow inactivation.

ABSTRACTVoltage-gated Na+ channels exhibit two forms of inactivation, one form (fast inactivation) takes effect on the order of milliseconds and the other (slow inactivation) on the order of seconds to minutes. While previous studies have suggested that fast and slow inactivation are structurally independent gating processes, little is known about the relationship between the two. In this study, we probed this relationship by examining the effects of slow inactivation on a conformational marker for fast inactivation, the accessibility of a site on the Na+ channel III-IV linker that is believed to form a part of the fast inactivation particle. When cysteine was substituted for phenylalanine at position 1304 in the rat skeletal muscle sodium channel (microl), application of [2-(trimethylammonium)ethyl]methanethiosulfonate (MTS-ET) to the cytoplasmic face of inside-out patches from Xenopus oocytes injected with F1304C RNA dramatically disrupted fast inactivation and displayed voltage-dependent reaction kinetics that closely paralleled the steady state availability (hinfinity) curve. Based on this observation, the accessibility of cys1304 was used as a conformational marker to probe the position of the fast inactivation gate during the development of and the recovery from slow inactivation. We found that burial of cys1304 is not altered by the onset of slow inactivation, and that recovery of accessibility of cys1304 is not slowed after long (2-10 s) depolarizations. These results suggest that (a) fast and slow inactivation are structurally distinct processes that are not tightly coupled, (b) fast and slow inactivation are not mutually exclusive processes (i.e., sodium channels may be fast- and slow-inactivated simultaneously), and (c) after long depolarizations, recovery from fast inactivation precedes recovery from slow inactivation.

Figure 3: Dose–response curve. Reaction rates for MTS-ET modification of F1304C are shown as a function of MTS-ET concentration used. All exposures were carried out at −120 mV and were either 50 (1 μM, n = 3; 2 μM, n = 6; and 4 μM, n = 10) or 25 (8 μM, n = 5) ms in duration. Rates were determined as described in Fig. 2. The dose–response curve was fit to an unweighted linear regression constrained to pass through the origin, giving a slope of 1.03 μmol−1 s−1.

Mentions:
To determine the degree of modification after each exposure, the average current between 40 and 42 ms after depolarization was divided by the corresponding value in the traces elicited from completely modified channels. A plot of fraction-modified versus cumulative exposure time was fit with a monoexponential rise that had a nonzero initial value reflecting the presence of persistent current before modification and a time constant whose reciprocal is the reaction rate between MTS-ET and cys1304 (Fig. 2 C). For reactions in 4 μM MTS-ET using 50-ms exposures at −120 mV, the reaction rate was 3.6 ± 0.3 s−1 (n = 10). We repeated this experiment at several different MTS-ET concentrations to test for a linear relationship between MTS-ET concentration and reaction rate, and thereby to confirm the presence of bimolecular kinetics (Fig. 3). A linear regression to this data yielded a slope of 1.03 μmol−1 s−1. Since F1304C has 38 cysteine residues, of which16 are believed to be intracellular, the presence of bimolecular reaction kinetics suggests that other cysteines are not involved in the MTS-ET-induced changes in the macroscopic current. All further experiments were performed either with 4 or 8 μM MTS-ET, both of which are within the linear portion of the concentration–rate curve.

Figure 3: Dose–response curve. Reaction rates for MTS-ET modification of F1304C are shown as a function of MTS-ET concentration used. All exposures were carried out at −120 mV and were either 50 (1 μM, n = 3; 2 μM, n = 6; and 4 μM, n = 10) or 25 (8 μM, n = 5) ms in duration. Rates were determined as described in Fig. 2. The dose–response curve was fit to an unweighted linear regression constrained to pass through the origin, giving a slope of 1.03 μmol−1 s−1.

Mentions:
To determine the degree of modification after each exposure, the average current between 40 and 42 ms after depolarization was divided by the corresponding value in the traces elicited from completely modified channels. A plot of fraction-modified versus cumulative exposure time was fit with a monoexponential rise that had a nonzero initial value reflecting the presence of persistent current before modification and a time constant whose reciprocal is the reaction rate between MTS-ET and cys1304 (Fig. 2 C). For reactions in 4 μM MTS-ET using 50-ms exposures at −120 mV, the reaction rate was 3.6 ± 0.3 s−1 (n = 10). We repeated this experiment at several different MTS-ET concentrations to test for a linear relationship between MTS-ET concentration and reaction rate, and thereby to confirm the presence of bimolecular kinetics (Fig. 3). A linear regression to this data yielded a slope of 1.03 μmol−1 s−1. Since F1304C has 38 cysteine residues, of which16 are believed to be intracellular, the presence of bimolecular reaction kinetics suggests that other cysteines are not involved in the MTS-ET-induced changes in the macroscopic current. All further experiments were performed either with 4 or 8 μM MTS-ET, both of which are within the linear portion of the concentration–rate curve.

Bottom Line:
In this study, we probed this relationship by examining the effects of slow inactivation on a conformational marker for fast inactivation, the accessibility of a site on the Na+ channel III-IV linker that is believed to form a part of the fast inactivation particle.We found that burial of cys1304 is not altered by the onset of slow inactivation, and that recovery of accessibility of cys1304 is not slowed after long (2-10 s) depolarizations.These results suggest that (a) fast and slow inactivation are structurally distinct processes that are not tightly coupled, (b) fast and slow inactivation are not mutually exclusive processes (i.e., sodium channels may be fast- and slow-inactivated simultaneously), and (c) after long depolarizations, recovery from fast inactivation precedes recovery from slow inactivation.

ABSTRACTVoltage-gated Na+ channels exhibit two forms of inactivation, one form (fast inactivation) takes effect on the order of milliseconds and the other (slow inactivation) on the order of seconds to minutes. While previous studies have suggested that fast and slow inactivation are structurally independent gating processes, little is known about the relationship between the two. In this study, we probed this relationship by examining the effects of slow inactivation on a conformational marker for fast inactivation, the accessibility of a site on the Na+ channel III-IV linker that is believed to form a part of the fast inactivation particle. When cysteine was substituted for phenylalanine at position 1304 in the rat skeletal muscle sodium channel (microl), application of [2-(trimethylammonium)ethyl]methanethiosulfonate (MTS-ET) to the cytoplasmic face of inside-out patches from Xenopus oocytes injected with F1304C RNA dramatically disrupted fast inactivation and displayed voltage-dependent reaction kinetics that closely paralleled the steady state availability (hinfinity) curve. Based on this observation, the accessibility of cys1304 was used as a conformational marker to probe the position of the fast inactivation gate during the development of and the recovery from slow inactivation. We found that burial of cys1304 is not altered by the onset of slow inactivation, and that recovery of accessibility of cys1304 is not slowed after long (2-10 s) depolarizations. These results suggest that (a) fast and slow inactivation are structurally distinct processes that are not tightly coupled, (b) fast and slow inactivation are not mutually exclusive processes (i.e., sodium channels may be fast- and slow-inactivated simultaneously), and (c) after long depolarizations, recovery from fast inactivation precedes recovery from slow inactivation.